Sampling method influences hemolymph bacterial communities in the eastern oyster, Crassostrea virginica

As the microbiome becomes increasingly recognized for its role in driving animal health and disease, accurate characterization of microbial community composition is essential. With this research comes the critical need for standardized sampling methods along with an understanding of the biases inherent to these methods. In oysters, hemolymph is a valuable system for microbiome research due to its diverse physiological functions, role as an indicator of health, and ease of collection. Unfortunately, much of the current literature regarding oyster hemolymph microbiome composition is opaque in its description of tissue collection methods. When these methods are clearly described, investigators often collect hemolymph via a needle inserted into the adductor muscle through a notch cut in the shell. However, due to the needle's movement through soft tissues and mucous membranes exposed to the surrounding seawater, this notch method carries significant risk of sample contamination. An alternative hemolymph sampling method, hereafter referred to as the hole method, minimizes this potential contamination as the needle is inserted through a hole drilled in the shell directly above the adductor muscle, circumventing other soft tissues. Here, we tested whether notch and hole hemolymph collection methods produce different 16S rRNA bacterial community profiles in C. virginica. To compare these two sampling methods and evaluate the potential method-specific biases, particularly biases that may result from tissue contamination, oysters had hemolymph collected through both a notch and hole while alternating method order. Following taxonomic classification and differential abundance analysis of the resultant 16S rRNA sequences, the presence of disproportionate Vibrio spp. enrichment in notch-associated samples was evident. Notch-collected samples yielded significantly higher 16S rRNA gene copy numbers (p = 0.037) and a 16-fold higher relative abundance of Vibrio spp. compared to hole-collected samples (52.6% vs. 3.2%, p _adj = 0.006) despite no significant change in absolute counts of the dominant genus Poseidonibacter. Overall, this analysis demonstrates both the potential of the hole sampling method and the need for microbiome researchers to account for biases in bacterial community composition associated with their chosen sampling method.